The group of Gram-positive bacteria that are generally referred to as lactic acid bacteria including Lactococcus spp. such as Lactococcus lactis, Lactobacillus spp., Streptococcus spp., Leuconostoc spp. and Oenococcus spp. are commonly used in the manufacturing of food products and feedstuffs, e.g. as dairy starter cultures in the manufacturing of fermented milk products such as butter, cheese and yoghurt. Lactococcus lactis is a typical example of a Gram-positive bacterium used for the manufacturing of a wide range of fermented milk products.
Besides, lactic acid bacteria are currently used as recombinant host cells for the production of heterologous and homologous gene products such as pharmaceutically active products or enzymes. Among the emerging industrial applications for L. lactis, recent work by the inventors has focused on the production of heterologous proteins with a potential as vaccines, therapeutics or enzymes. The expression system used includes a strong regulated promoter and has allowed a high-level production of recombinant Leuconostoc mesenteroides β-galactosidase, LacLM (Madsen et al., 1999).
With the development of microorganisms as cell factories for the production of heterologous proteins, a number of genetic tools for improved gene expression have been established. These include strong promoters, high copy number vectors, optimised codon usage and improved production strains, and their use has resulted in an increase of production levels.
Using these optimised tools, secretion of heterologous proteins into the culture supernatant might represent a limiting step. Therefore, the molecular knowledge of protein secretion is also emerging as a subject of applied research. To facilitate downstream processing of recombinantly produced protein, secretion of the protein is generally desired. To achieve efficient secretion of heterologous gene products it is required that constructs are used in which the gene coding for the desired gene product is operably linked to a gene coding for an effective signal peptide that can be recognised by the signal peptidases of the host cell.
The process of secretion in bacteria includes events that occur just after translation of the mRNA, i.e. the subsequent recognition of the signal peptide (SP) in the nascent unfolded polypeptide chain by the Sec apparatus and cleavage by signal peptidase upon translocation through the cell membrane.
The Sec-dependent pathway is the best studied system for protein export. Although it is known that virtually all proteins exported via this mechanism require a SP, it is not clearly understood how the structure of the SP interacts with the different components of the secretion machinery in the cell. The recent characterization of a Sec-independent pathway that is conserved between E. coli and plants illustrates the fact that proteins are exported through a number of distinct pathways (Settles and Martienssen, 1998; Stephens 1995). The mechanisms involved normally require the presence of sequence motifs in the exported protein.
SPs are the N-terminal extensions present in Sec-dependent secreted proteins. The structure of a typical SP includes three distinct regions: (i) an N-terminal region that contains a number of positively charged amino acids, lysine and arginine; (ii) a central hydrophobic core and; (iii) a hydrophilic C-terminus that contains the sequence motif recognised by the signal peptidase (von Heijne 1990). Despite structural similarities, large sequence variation is observed between different SPs. This variation has recently been related to specific targeting of the secreted proteins (Martoglio and Dobberstein, 1998). Studies of secretion in Escherichia coli have shown the influence of the hydrophobic core region of SP on efficient processing. SPs with highly hydrophobic core regions supported a high rate of transport even when an altered N-terminal region with negative charge is used (Izard et al., 1996). PhoA has been used as a model protein for detailed secretion studies in this bacterium. The effect of the removal of helix-breaking residues (Gly or Pro) can be compensated by increased hydrophobicity (Izard et al., 1995). In competition experiments, two identical SPs were placed N-terminal to PhoA and the rate of utilization of either SP was shown to be dependent on small increases in the hydrophobicity of one of the SP (Chen et al., 1996). Moreover, it was shown that a reduced negative charge at the amino terminus resulted in a lower overall affinity for the transport pathway (Izard et al., 1996).
The characterisation of numerous extracellular proteins has allowed development of a method for the prediction of the presence and location of signal peptide cleavage sites in amino acid sequences from different organisms including Gram-positive and Gram-negative prokaryotes, and eukaryotes (Nielsen et al., 1997). The method involves a prediction of cleavage sites and a signal peptide/non-signal peptide prediction based on a combination of several artificial neural networks. The use of this method permits the preliminary design and analysis of SP derivatives prior to their construction and test in vivo.
Proteins that are targeted for secretion include a signal sequence or signal peptide (SP) at the N-terminus. SPs are recognised and cleaved by a leader or signal peptidase, a component of the secretion machinery of the cell, during translocation across the cell membrane (Martoglio and Dobberstein, 1998). SPs are normally 25 to over 35 amino acids (aa) in size in Gram-positive bacteria. SPs do not share sequence homology, but are often composed of an amino terminus that includes one or more basic aa, a central hydrophobic core of seven or more aa, and a hydrophilic carboxy terminus containing the motif that is recognized by signal peptidases (Martoglio and Dobberstein, 1998). A survey of available SPs from L. lactis suggested the use of the SP from Usp45, the major secreted lactococcal protein (van Asseldonk et al., 1990). This SP was reported to be functional in the secretion of several heterologous proteins in L. lactis (van Asseldonk et al., 1993).
Traditional strategies for the identification of SPs in L. lactis have followed the construction of genomic libraries in a vector carrying a promoterless reporter gene. In general, work in Gram-positive bacteria has involved the use of reporter genes with a demonstrated functionality for the identification of SPs in Gram-negative bacteria. These reporters include BlaM, the E. coli β-lactamase (Sibakov et al., 1991; Perez-Martinez et al., 1992). The use of BlaM for the identification of L. lactis SPs implied a limitation on the possibility of direct screening in L. lactis and this has been assumed to be due to differences in codon usage and protein folding requirements for BlaM (Pouquet et al., 1998). Therefore, primary screening of Gram-positive bacterial genomic libraries has up till now been carried out in E. coli and positive clones subsequently tested in L. lactis (Sibakov et al., 1991; Perez-Martinez et al., 1992) thereby imposing a tedious and labour consuming selection step for functionality in the primary host. A more appropriate secretion reporter, the extracellular β-amylase from Bacillus licheniformis has also been used in screening for lactococcal SPs, but following the same screening strategy (Perez-Martinez et al., 1992). These strategies resulted in the isolation of SPs of type I exclusively. Moreover, some of the functionality of the sequences identified was due to the presence of amino acid residues derived from the multiple cloning site in the vector. These amino acids matched the requirements for the C-terminal region of this type of SPs (Perez-Martinez et al., 1992).
However, it is desirable to dispose of a broad range of SPs in order to select, for specific purposes, such SPs that are suitable in a particular host cell or for the secretion of a particular gene product. A major objective of the present invention is therefore to provide a convenient method for direct isolation of lactic acid bacterial nucleotide sequences coding for signal peptides that are functional in a broad range of host cells including lactic acid bacterial cells, which method does not require an intermediate screening step in another species. A further objective of the invention is to provide a transposable element useful in the present method that permits to identify and locate, in a bacterial chromosome, sequences coding for SP. By using the novel method several novel lactococcal SPs were identified, isolated and improved by mutagenesis.